Patients

Ten patients from the liver transplant program of the Santo António General Hospital were included in the study. These 10 patients were chosen out of a cohort of 34 patients. Selection criteria were that all patients had one positive antigenemia determination and half of them had clinical signs of CMV disease. Seven of the patients were males and 3 were females. Patients' ages ranged from 29 to 58 years, with an average of 38.5 years. One patient was transplanted due to the presence of alcoholic cirrhosis and 9 due to Familial Amiloidotic Polyneuropathy (FAP). Of these 8 had the Met 30 mutation, and 1 the Pro52 mutation. The immunosupression protocol included 500 mg metilprednisolone at the beginning of the surgery and at the anehepathic phase, followed by 4 doses of 50 mg on the first day post-transplant, 4 doses of 40 mg on the second day, 4 doses of 30 mg on the third day, 4 doses of 20 mg on the 4th day, 2 doses of 20 mg on the 5th day, and one dose of 20 mg on the 6th day. Thereafter a dose of 20 mg/day was administered until the end of the 3rd month, 15 mg/day until the end of the first year, 10 mg/day until the end of the 18th month, 5 mg/day until the end of the 2nd year, after which immunosupression was discontinued. The protocol also included the administration of 50 mg azatioprin during the anhepathic phase followed by 50 mg every 12 h during 30 days. Neoral Ciclosporin was also administered, and the drug level monitored by the Tdx method. Drug levels were kept at 350-450 ng/ml during the 1^st month post-transplant, 250-350 ng/ml during the second and third months, 150-250 ng/ml until the end of the first year, and 75-150 ng/ml thereafter. Acute cellular rejection was treated with 1 g metilprednisolone for 3 days and thereafter the reduction scheme followed on the post-transplant was applied.

All patients were monitored for the presence of an active infection of CMV, starting between the 1st and 2nd week post-transplant, weekly during the first month and later at each clinical appointment (one and 3 weeks after disclosure and monthly thereafter), or whenever it was clinically relevant. A total of 87 samples were collected from all 10 patients with an average of 8.7 samples per patient (range of 4 to 12 samples). Twin blood samples were collected on sterilized EDTA tubes. One of the samples was prospectively tested by pp65 antigenemia and the result was used for clinical management. The other sample was retrospectively studied by molecular biology methods. Scarcity of the available sample did not allow CMV pp67 determination on 5 specimens.

Two of the 10 patients were retransplanted. One of these was retransplanted 24 h after the first transplant due to the presence of a liver arterial thrombosis. The other patient, presented multiple liver abscesses, was retransplanted twice (3 and 7 weeks after the first transplant) and died during the second retransplantation for reasons not related to CMV infection.

Nine of the 10 patients were transfused with leucodepleted red blood cells.

Nine of the patients were CMV positive before transplant, and the remainder patient was CMV negative.

All liver donors were CMV Ig positive.

CMV infection definition

CMV infection is defined as isolation and/or detection of virus from any tissue or body fluid or serological conversion to CMV in a patient who was seronegative before transplantation. In the context of the present work, we used antigenemia as the golden standard method for CMV detection, and consequently CMV infection definition.

CMV disease definition

CMV disease is usually defined as CMV infection accompanied by clinical manifestations. The most common of these is a viral syndrome with malaise, fever, leucopenia, atypical lymphocytosis, thrombocitopenia, mild to moderate elevations of serum aminotransferases often in a self-limiting process that does not require treatment. Of greater concern is organ involvement that may be localized to a single organ or disseminated if there is involvement of 2 or more non-contiguous organ sites. Organ involvement is defined as histopathologic evidence of CMV with or without a positive viral culture of involved tissue.

In the context of the present work, CMV disease was defined as presence of the above mentioned clinical signs in association with positive pp65 antigenemia. Whenever this happened, ganciclovir therapy (5 mg/Kg every 12 hours) was always initiated for 14 days, or until negative antigenemia results were observed. Thus we considered all patients who were not treated as without CMV disease. For the same reason, the moment of ganciclovir prescription was considered as the onset of CMV disease.

Antigenemia pp65

CMV antigenemia assay was carried out on 3 ml of EDTA-anticoagulated blood within 4 hours of specimen collection. The pp65 antigen was detected with the commercial assay CMV-vueTM (DiaSorin, Stillwater, Minnesota 55082-0285, USA), with a few modifications. The antigenemia assay consists of four major steps - isolation of the polymorphonuclear leukocytes from 3 mL of EDTA-anticoagulated blood by dextran separation and ressuspension of the leukocyte in PBS to give a final cell density equivalent to 2 × 10⁶ leukocytes/mL; preparation of two cytospin slides and fixation of the cells in formaldehyde; incubation with a mix of two mouse monoclonal antibodies (C10 and C11) directed against the pp65 followed by incubation with horseradish peroxidase-coupled rabbit anti-mouse IgG after which a peroxidase mediated chromogenic reaction takes place; quantification under a light microscope by counting the number of positively stained leukocytes and the total number of leukocytes per slide. The results were given as the total number of positive leukocytes/50.000 leukocytes.

Cobas amplicor CMV monitor

For quantification of CMV DNA, the commercial assay Cobas Amplicor CMV Monitor Test (Roche Diagnostics Systems, Inc., Branchburg, NJ, USA), directed against the CMV polymerase gene, was performed on 200 μl of plasma according to the manufacturer recommendations (see ref. 12 for details). The results were expressed as DNA copies per mL of plasma. The linear range of the assay is 400-400.000 copies of CMV DNA per mL.

Nuclisens CMVpp67

Amplification and detection of CMV pp67 mRNA transcripts was done on 100 μl EDTA anti-coagulated blood using the commercial assay Nuclisens pp67 Assay (Organon Teknika, Boxtel, Netherlands) according to the manufacturer's instructions (see 16 for details).

Total nucleic acids were isolated by the Boom guanidinium thiocyanate-silica procedure in a semi-automated procedure [18] with Nuclisens Automated Isolation Reagents (Organon Teknika, Boxtel, Netherlands) and Nuclisens^® Extractor (Organon Teknika).

The results were expressed as presence or absence of CMV pp67 mRNA.

Statistical analysis

The performance of the 3 methods used, relative to the prediction of the development of disease, was statistically analysed using the following definitions: Sensitivity was calculated as the proportion of the determinations done during disease episodes that tested positive; Specificity was defined as the proportion of the determinations done during disease-free episodes that tested negative; Positive predictive value was calculated as the proportion of "test-positive" determinations that occurred on patients that developed disease; Negative predictive value was defined as the proportion of "test-negative" determinations that occurred on patients that did not develop disease.

Determinations on patients that developed disease were compared with the results observed on assymptomatic patients using the Mann-Whitney U test.

Overall test performance

All 87 sequential specimens obtained from 10 liver transplant recipients were assessed by CMV antigenemia and CMV monitor, whereas only 82 specimens were available for CMV pp67 mRNA assessment. The presence of an internal control in CMV monitor assay allowed the identification of 1/87 samples with amplification inhibitors. A similar control was also present in the Nuclisens CMV pp67 assay, revealing that none of the samples studied showed signs of reaction inhibition. The results obtained with the 3 tests were concordant in 58/82 samples (70%). Of these, 57% (33/58) were negative and 43% (25/58) were positive for CMV.

Taking the antigenemia determination as the "golden standard" (the routine method used for clinical management), all patients developed CMV infection. According to the serostatus before transplantation, 1 patient had a primary CMV infection. CMV monitor test detected CMV infection in 10/10 patients, while CMV pp67 mRNA was only capable of detecting the infection in 8/10 patients. It must however be noted that the two discordant patients never developed CMV disease.

The onset of CMV infection was on average 31 days (range 17-69) by antigenemia pp65, 40 days (range 23-70) by CMV monitor, and 37 days (range 17-69) by pp67 mRNA assay. Contrary to our expectations, CMV positivity was never detected earlier by molecular biology assays than by antigenemia. In fact, the 4 patients with discordant ascending kinetics (patients A, B, D and E - Fig 1) presented a positive antigenemia result one determination earlier than the two other assays. It must however be noticed that: 1) two of the 4 patients had no CMV pp67 determination at the discordant points; 2) antigenemia determination was in all cases only 1 positive cell/50,000 cells, and; 3) none of the 4 patients ever developed CMV disease.

CMV pp67 mRNA assay was found to show positivity latter than CMV monitor, since 3 patients (A, D and E - Fig 1) had positive antigenemia, positive CMV monitor and negative pp67 mRNA. Again it must be noted that none of these patients ever developed CMV disease and CMV pp67 mRNA assay was always negative for patients D and E (fig 1). Considering CMV clearance as one negative result by at least one CMV viremia assay, after being positive by the same assay, 8 patients (all but patients D and F - fig 1 and 2) showed analytical signs of CMV clearance. In these patients, antigenemia was found to be the first test to become negative in 3/8 patients (H, I, J-fig 2). Furthermore, this method was found to become negative concomitantly to Nuclisens CMV in 3/8 patients (B, C and I - Fig 1 and 2), whereas CMV monitor was the last test to show CMV clearance in 4/8 patients (B, C, H, I-fig 1 and 2).

Correlation of analytical results to CMV disease

Only 5/10 patients developed clinical CMV disease, as defined in the Materials and Methods section. On average, the onset of CMV disease was 36 days post-transplant (range 30-43 days), and at this time all tests showed positive results (fig 2).

In order to identify the most sensitive method for the identification of patients at risk of developing CMV disease, we compared results obtained on the available samples before the onset of disease (one week on average; range 4-15 days). As can be seen on table 3, the results showed that if we set an antigenemia cut of value of 8/50,000 cells, patients with disease had antigenemia results higher than the observed for patients without disease. The only exception among patients with disease was patient J, that showed negative determinations 15 days before the onset of clinical disease, and no latter determination was available before disease was observed. Also, patient C, despite not developing CMV disease showed one determination with 31 cells/50,000. Curiously, at this stage, all methods showed positive values.

Finally, when taking in consideration all determinations, the 3 methods seemed to behave similarly (table 4), again, with slightly better raw data performance observed for the pp67 mRNA assay.